Future oilcrops for a demanding world

Oil-producing crops currently occupy about 10% of global arable land and are second only to carbohydrate crops in terms of their importance as providers of calories for both humans and their livestock. In addition to their edible roles, oil crops also provide a wide range of industrial products, known collectively as oleochemicals, and are increasingly used as a biofuel, especially biodiesel, and as feedstock by the chemical industry.Due to the increasing utilization of these plant products, a substantial increase in the total production of vegetable oil is required. This increase has the potential to be met by increasing the oil content in presently used oil crops or introducing new high-oil-yielding crops. However in order to increase the yield of lipids in plants, the pathway that produces these compounds and the mechanisms that control it must be better understood. Most plants accumulate oil in the seed predominantly in the form of triacylglycerols (TAGs), a glycerol backbone onto which three fatty acids are sequentially esterified. The synthesis and assembly of TAG in plants is complex, involving a metabolic network of fatty acid fluxes through multiple subcellular compartments containing alternative pathways to produce different lipid compositions. Much progress has been made in understanding how plants produce and accumulate oils. The specific enzymes involved in the metabolic pathway leading to triacylglycerols (TAGs) stored in the oil bodies, as well as the pathway that supplies the precursors generated from imported sucrose, are to a large extent known. However, we still have a poor understanding in key areas such as factors important for regulating the flux of photosynthates into storage compartments, the synthesis of fatty acids, or the level of oil content in storage tissues. Hence, research in these areas is of great importance to enable a substantial increase in vegetable oil production

Oils and fats on food: is it possible to have a healthy diet?

Oils and fats are an important part of our diet as components of many food formulations. Thus, they are retailed for domestic or hostelry uses and broadly used by food industry for the elaboration of margarines, ice cream, canned food, pre-cooked dishes, bakery, confectionary, chocolates, etc. Chemically, the main component of oils and fats are triacylglycerols (TAGs), which account for up to 95% of their total weight. They consisted of a molecule of glycerol esterified with three fatty acids, usually the saturated, palmitic and stearic, the monounsatu�rated oleic, and the polyunsaturated, linoleic or linolenic, all with 18 carbons excepting the palmitic which has 16 carbons. Out of those most common fatty acids, we can found other fatty acids present only in certain oils such as saturated medium chained fatty acids like lauric and myristic, which contain 12 and 14 carbons respectively

Oils and fats on food: is it possible to have a healthy diet?

Oils and fats are an important part of our diet as components of many food formulations. Thus, they are retailed for domestic or hostelry uses and broadly used by food industry for the elaboration of margarines, ice cream, canned food, pre-cooked dishes, bakery, confectionary, chocolates, etc. Chemically, the main component of oils and fats are triacylglycerols (TAGs), which account for up to 95% of their total weight. They consisted of a molecule of glycerol esterified with three fatty acids, usually the saturated, palmitic and stearic, the monounsatu�rated oleic, and the polyunsaturated, linoleic or linolenic, all with 18 carbons excepting the palmitic which has 16 carbons. Out of those most common fatty acids, we can found other fatty acids present only in certain oils such as saturated medium chained fatty acids like lauric and myristic, which contain 12 and 14 carbons respectively

New Insights Into Sunflower (Helianthus annuus L.) FatA and FatB Thioesterases, Their Regulation, Structure and Distribution

Sunflower seeds (Helianthus annuus L.) accumulate large quantities of triacylglycerols (TAG) between 12 and 28 days after flowering (DAF). This is the period of maximal acyl-acyl carrier protein (acyl-ACP) thioesterase activity in vitro, the enzymes that terminate the process of de novo fatty acid synthesis by catalyzing the hydrolysis of the acyl-ACPs synthesized by fatty acid synthase. Fatty acid thioesterases can be classified into two families with distinct substrate specificities, namely FatA and FatB. Here, some new aspects of these enzymes have been studied, assessing how both enzymes contribute to the acyl composition of sunflower oil, not least through the changes in their expression during the process of seed filling. Moreover, the binding pockets of these enzymes were modeled based on new data from plant thioesterases, revealing important differences in their volume and geometry. Finally, the subcellular location of the two enzymes was evaluated and while both possess an N-terminal plastid transit peptide, only in FatB contains a hydrophobic sequence that could potentially serve as a transmembrane domain. Indeed, using in vivo imaging and organelle fractionation, H. annuus thioesterases, HaFatA and HaFatB, appear to be differentially localized in the plastid stroma and membrane envelope, respectively. The divergent roles fulfilled by HaFatA and HaFatB in oil biosynthesis are discussed in the light of our data.España MINECO y FEDER Projects AGL2014- 53537-R y AGL2017-83449-

Improvement of nutritional properties of Cassva (Manihot esculenta) through massive analysis of gene expression

Motivation: Currently, the cassava is the basis of food for more than 1 billion people in the world. In this instance, the modification of the nutritional composition of plant foods is an urgent matter, since the basic nutritional needs of the world population are not yet covered. The identification of the transcription factors that regulate oil biosynthesis could give tools to re-direct sucrose to oil in the root of tuberculous cultures. In cassava, most of the research that has been done has focused on the tuber, with little study of the seeds and their characteristics.In Cassava the factors that direct the flow of carbon towards the different reserve tissues and determine the final composition of the tissue in the plant are not known. The proposed research line aims to deepen the knowledge of the fatty acid biosynthesis in cassava and increase the oil content of the roots. Fundamental knowledge to be able to reach the final objective of increasing the oil content. In addition to improving its nutritional value, while the amount of nutrients that contribute to the body.Methods: There are two lines of research. The computer methods are based on searching for genes involved in the biosynthesis pathway of lipid substrates through databases such as Uniref and UniProt. After a series of genes obtained from other trials, we compared their presence in the Manihot Esculenta genome and verified their function. With homologous genes, we can expand the number of candidate genes.The laboratory methods aim to develop the method of genetic transformation of cassava (Ima M Zainuddin et al., Plant Methods 2012, 8:24) in combination with Agrobacterium. From a horizontal cuttings and an in vitro culture of their apical buds, we get a mother plant from which we can obtain somatic embryos and create new seedlings to be able to transform with differentResults: The following results were obtained after a first search for genes obtained from a compilation base don papers previously made in other related organisms. From that list of candidates genes, there are genes that cease to be candidates because they are not found in Manihot esculenta, these being susy, ATP-PFK and FBAGenes that continue as candidates, for being in the genome of cassava, being PK,GPT2 and PPT1. And for the latter, new genes can be obtained, such as APE2 with identifier 16G010700.1 in JGI Phytozome. It is a translocator of triose-phosphate located in the chloroplast.Conclusions: There is a vision of the future in this line of research to be able to start from a new and broader list of genes involved in the synthesis routes of sucrose and oil that are susceptible to modification.There is still a lot to study to reach the goal, but positive results will come out

Nutritional improvement of Manihot esculenta roots by boosting the lipids storage

Motivation: The human population continues to grow and it is necessary to produce more and better quality food to meet the world population's demand. Genetic engineering opens great possibilities to improve the quantity of available food. The cassava (Manihot esculenta) plant is the basis of food for more than 1 billion people in the world. All plants have genes coding for oil biosynthetic pathways and transcription factors that activate the expression of these genes. If these transcription factors are activated in other tissues, like roots, the conversion of sucrose to oil could increase. In this way, crops that accumulate sugars and starch could become more nutritious (2.2 times more energy than carbohydrates). It has been shown that starch and oil can accumulate in the same cell, as is the case for oats (Ekman et al., 2008). In this project, cassava has been chosen as a model plant because it has a high starch content in their edible roots.Methods: Two somatic embryos were obtained from mother plants with ecotype 60444. The in-vitro plants will be transformed by the vector via Agrobacterium tumefaciens. The functional annotation of the cassava proteome was carried out using Sma3s (Casimiro-Soriguer et al., 2017). This annotation will allow us to know the function of the protein-coding genes present in cassava. To know those genes involved in the synthesis of fatty acids they must be filtered. The expression of these genes in different tissues was comparated with ArrayExpress. Posible candidates will be examined in order to choose the most suitable ones to be transformed and expressed in the cassava plants.Results: From 41,381 cassava predicted proteins, 35,889 were scored, meaning Sma3s annotated 86% of the proteome. The list of possible candidates is currently around 600 genes and their expression wilI checked with public database. In vitro plants are growing and the second phase of the transformation will be begun.Conclusions: The project will (i) expand knowledge on cassava, particularly on the development of their storage organs, such as roots and seeds, as well as carbohydrate and lipid metabolism, and (ii) develop a cassava crop modification platform using genetic engineering techniques. This work aims to cover two demands of society, try to mitigate hunger, and on the other hand be able to extrapolate the scientific knowledge generated to other crops of interest to cover the current demand for vegetable oil

CRISPR/Cas9 mediated genome edition in castor plant based on Golden Gate Assembly

Motivation: CRISPR/Cas9 technology has been developed as the most efficient and widely used genome editing tool to modify genomes of numerous plants, where cas9 cuts in the double strand of DNA is driven by a sequence of 20 nucleotides included in a single guide RNA (sgRNA). However, simultaneous editing of multiple targets using CRISPR/Cas9 remains a technical challenge in this field (Ma et al., 2014).Methods: In the present work, Golden Gate Assembly cloning strategy was used to generate multiple CRISPR/cas9 editing structures to be used in castor plant. Modular cloning systems use type IIS enzymes to cut outside their recognition site allowing efficient assembly of DNA fragments with compatible overhangs, facilitating the correct orientation of multiple sequences simultaneously (Engler et al., 2014). Our main goal was obtaining a genetic construction that allows the expression of two sgRNAs together with the cas9 nuclease in the same plasmid vector for transformation of castor via Agrobacterium infection.Two CRISPR targets for FAH12 castor hydroxylase were selected to avoid possible off targets. These targets were included in sgRNAs and cloned in level 0 plasmids, each one flanked by restriction sites for the BsaI enzyme. Golden Gate level 1 reaction includes several BsaI digestion and ligation cycles that assemble U6 promoter with both sgRNAs separately into level 1 plasmids flanked by BpiI restriction sites. Simultaneously, cas9 enzyme was cloned under the control of a double strong 35S promoter followed by the nopaline synthase (nosT) terminator from level 0 plasmid including those elements into another level 1 plasmid, also flanked by BpiI restriction sites. Digestion with BpiI then show compatible overhangs in all of level 1 elements (U6-sgRNA1, U6-sgRNA2, 2x35S-cas9-nosT), which can be assembled in correct order and orientation into a level 2 structure. The final result was a level 2 plasmid including all the elements required for CRISPR/cas9 multiplex genome editing of FAH12 hydroxilase. This construction will be transferred to Agrobacterium for later castor embryos transformatio

High stearic sunflower oil: Latest advances and applications☆

Regular sunflower oil is rich in linoleic acid. To improve its properties for different applications several genotypes with modified fatty acid compositions have been developed. Amongst them, the most remarkable have been high oleic and high stearic types. High stearic sunflower lines reported to date have been produced by traditional methods of breeding and mutagenesis. The mutations affected the expression of enzymes responsible for stearate desaturation in developing seeds. This trait has been combined with standard and high oleic backgrounds, giving high stearic lines with high contents of linoleic or oleic acids and thus different physical properties, increasing their functionality and potential applications. Nevertheless, for applications requiring plastic or confectionery fats, the oils have to be fractionated to obtain derived fats and butters with higher levels of solids. In the present review we present recent advances for the above mentioned topics related to high stearic sunflower oils

A metabolomic perspective of the impact of mitochondrial prohibitin on C. elegans longevity.

The mitochondrial prohibitin complex is a context-dependent modulator of longevity. Specifically, prohibitin deficiency shortens the lifespan of otherwise wild type worms, while it dramatically extends lifespan under compromised metabolic conditions, as in the case of the diapause daf-2(e1370) mutant. This extremely intriguingly phenotype has been linked to alterations in mitochondrial function and in fat metabolism. Nevertheless, the true function of the mitochondrial prohibitin complex remains elusive. With the ultimate goal of understanding how mitochondrial prohibitin complex affects longevity, we have employed several metabolomic approaches to characterize the changes elicited upon prohibitin depletion by RNAi on the metabolome of wild type and daf-2 mutant worms.Metabolic analysis by gas chromatography coupled to a flame ionization detector and 1 H-NMR spectroscopy reveals that prohibitin depletion leads to an alteration in the overall fatty acid composition of the worm, as well as in carbohydrate and amino acid metabolism. To enlarge the coverage of the metabolome, we employed a lipidomic mass spectrometry-based approach. We identify that prohibitin has a differential effect in the content of various species of triglycerides and phospholipids in wild type and in daf-2 mutant animals. In particular, we find that prohibitin affects not only the amount but also the composition of fat storage lipids. Overall, prohibitin depletion has a more pronounced effect on the metabolic profiles of wild type worms than of daf-2 mutants indicating that daf-2 mutants are more robust to the changes elicited upon prohibitin depletion. We are currently exploring the relevance of identified metabolites in the context of the effect of prohibitin on the C. elegans longevity.Peer Reviewe

Molecular cloning and biochemical characterization of three phosphoglycerate kinase isoforms from developing sunflower (Helianthus annuus L.) seeds

Three cDNAs encoding different phosphoglycerate kinase (PGK, EC 2.7.2.3) isoforms, two cytosolic (HacPGK1 and HacPGK2) and one plastidic (HapPGK), were cloned and characterized from developing sunflower (Helianthus annuus L.) seeds. The expression profiles of these genes showed differences in heterotrophic tissues, such as developing seeds and roots, where HacPGK1 was predominant, while HapPGK was highly expressed in photosynthetic tissues. The cDNAs were expressed in Escherichia coli, and the corresponding proteins purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Despite the high level of identity between sequences, the HacPGK1 isoform showed strong differences in terms of specific activity, temperature stability and pH sensitivity in comparison to HacPGK2 and HapPGK. A polyclonal immune serum was raised against the purified HacPGK1 isoform, which showed cross-immunoreactivity with the other PGK isoforms. This serum allowed the localization of high expression levels of PGK isozymes in embryo tissues.Ministerio de Ciencia e Innovación AGL2011-2318